The long-term objective of this project is to understand the relationship between the molecular structure of the mitochondrial citrate transport protein (CTP) and its mechanism of transport. This transporter catalyzes the exchange of tricarboxylates, dicarboxylates, and phosphoenolpyruvate across the inner mitochondrial membrane, and as such is essential to the energy metabolism of eukaryotic cells. Recently, we: i) conducted cysteine scanning mutagenesis studies of transmembrane domains (TMDs) HI and IV which, in combination with chemical modification, nitroxide scanning, and substrate protection experiments, permitted identification of essential portions of the substrate translocation pathway; ii) developed a homology model of the CTP structure; and iii) developed methods for the purification of the CTP in crystallization-compatible detergents, which enabled the initiation of comprehensive crystallization trials. From this foundation, we propose to launch studies that will continue the fundamental advancement in our understanding of the functioning of this metabolically important transporter. Specifically, experiments will be conducted to: i) define the contributions of the four remaining TMDs in the formation of the CTP substrate translocation pathway (via cysteine-substitution mutagenesis at locations chosen on the basis of our homology modeled CTP structure followed by chemical modification of the single Cys mutants) and identify residues forming an electrostatic funnel that attracts citrate into the pathway from its surfaces; ii) identify the substrate binding site(s) within the translocation pathway and assess the ability of selected CTP domains to control substrate access to the pathway; iii) identify residues forming the interface between two CTP monomers in homodimeric CTP and characterize the ligand-induced conformational changes that occur during transport using site-directed spin labeling and thiol cross-linking; and iv) identify conditions enabling the growth of X-ray diffraction quality CTP crystals followed by determination of the CTP structure. These studies will provide a comprehensive understanding of the chemical and structural bases for mitochondrial CTP function. The health relatedness of this project concerns the central role of the CTP in bioenergetics. Thus, altered CTP function in disease (e.g., diabetes, cancer) is an important aspect of the aberrant metabolism that characterizes these pathologies. Consequently, an elucidation of the structural basis for substrate transport through the CTP is critical to understanding the CTP's role in energy production in normal and pathological states.